Paavo Kalo

Analysis of free and esterified sterols in fats and oils by flash chromatography, gas chromatography and electrospray tandem mass spectrometry.

A method for the analysis of free and esterified sterols has been developed. Fat or oil samples were separated on solid-phase extraction silica gel columns into a sterol ester fraction, a fraction of triacylglycerols, and a free sterol fraction containing partial acylglycerols and residual triacylglycerols. Sterol esters and acylglycerols of the free sterol fraction were transesterified to methyl esters. The fatty acid methyl esters from sterol ester fraction and the free sterols from sterol ester fraction and free sterol fraction were determined by GLC. Precursor ion electrospray MS-MS of sterol fragment ions of sterol ester fractions were recorded and used for determination of sterol ester proportions in butterfat and vegetable oil samples.

Regiospecific determination of short-chain triacylglycerols in butterfat by normal-phase HPLC with on-line electrospray-tandem mass spectrometry.

This study uses normal-phase HPLC with on-line positive ion electrospray mass spectrometry (ESI-MS) to obtain quantitative compositional data on both synthetic and butterfat short-chain TAG. The product ion tandem MS of standards averaged 11.1 times lower in abundance of the ion formed by cleavage of FA from the sn-2-position for the pairs of regioisomers in the TAG classes: L/L/S-L/S/L and L/S/S-S/L/S, where L denotes long and S short acyl chain (C2−C6). The molar correction factors, determined for 42 regioisomeric pairs of short-chain TAG of 20 randomized mixture of standards, differed by 1.4–80% as the ratios varied between 0.217 and 5.847. Butterfat TAG were resolved into four fractions on short flash chromatography grade silica gel columns. Pairs of regioisomers in the TAG classes L/S/S-S/L/S with predominance of L/S/S isomers and the sole regioisomers in the TAG classes L/L(M)/S were identified by tandem MS, where M denotes either 8∶0 or 10∶0 acyl chain. The total proportion of L/L(M)/S isomers was estimated at 34.7 and that of L/S/S-S/L/S at 1.0 mol%, including a small proportion of S/S/S. In contrast to previous work, the present data indicate the presence of a small proportion of butyric and caproic acids in the sn-1-position. The overall distribution of the FA in the short-chain TAG of butterfat, calculated from direct MS measurements, was consistent with the results of indirect determinations based on stereospecific analyses of total butterfat.

Chromatographic methods in the monitoring of lipase-catalyzed interesterification

Structured lipids containing special fatty acids were produced via lipase-catalyzed interesterification. Chromatographic methods were modified for the analysis of the acylglycerol content of the selected interesterified products, resulting from different reacting substrate mixtures and reaction systems. A modified normal-phase high performance liquid chromatographic method with evaporative light-scattering detection was used in the analysis of the diacylglycerols formed as intermediate products during interesterification. One of the advantages of the method was the low solvent consumption through the use of a narrow-bore silica column. Another advantage was in the speed of the analysis. Using a suitable solvent gradient, both diacylglycerol and triacylglycerol components were separated by reversed-phase high performance liquid chromatography within 50 min, providing both the diacylglycerol content and the triacylglycerol composition. High-temperature gas chromatography was also used to separate both diacylglycerol and triacylglycerol molecular species after trimethylsilylation of the structured lipid samples. Good agreements in the values obtained for the concentrations of diacylglycerols and the triacylglycerol compositions were apparent with the different chromatographic methods.

Mass spectrometric identification of triacylglycerols of enzymatically modified butterfat separated on a polarizable phenylmethylsilicone column

Original butter oil, butter oil interesterified withPseudomonas fluorescens lipase as catalyst, and the saturated, monoene, diene and triene fractions of interesterified butter oil from silver ion chromatography on ap-propylbenzene sulfonic acid column were analyzed on a polarizable phenyl(65%)methylsilicone column. Major and also some minor molecular species of triacylglycerols (TG) were identified from the electron-impact (EI) gas chromatography/mass spectrometry analytical data for the original and lipase-modified butter oil and the four fractions. Acyl + 74 and acyl + 128 fragments of EI mass spectral data proved useful, in addition to acyl and M - RCOO fragments, in the identification of molecular species of TG. The TG compositions of the modified butter oils were compared with the composition calculated according to random distribution and with the TG composition of original butter oil.

Determination of positional distribution of butyryl groups in milkfat triacylglycerols, triacylglycerol mixtures, and isolated positional isomers of triacylglycerols by gas chromatography and1H nuclear magnetic resonance spectroscopy

Positional isomers (1-butyryl-2X-3Y-rac-glycerol and 2-butyryl-1X-3Y-rac-glycerol;X,Y=long-chain acyls) of saturated triacylglycerols (TAG) with 34 and 40 acyl carbons were shown to separate in two chromatographic peaks on immobilized phenyl(65%) methylsilicone column by gas-liquid chromatography, and on reversed-phase ODS-1 column by high-performance liquid chromatography. The analysis of 500-MHz1H nuclear magnetic resonance (NMR) spectra showed distinct differences between 2-butyryl-1X-3Y-rac-glycerol and 1-butyryl-2X-3Y-rac-glycerol isomers in the resonance signals of methylene and methine protons of glycerol backbone, and carbon-2 methylene of acyl groups, and methyl protons of butyryl group. The1H NMR spectra of three interesterified mixtures of three monoacid TAG containing saturated butyrate and caproate TAG and unsaturated butyrate TAG showed that triplets of methyl protons of butyryl groups atsn-1(3)- andsn-2-positions in saturated and unsaturated TAG had similar chemical shifts and that the chemical shift of caproyl methyl protons was different from those of butyryl methyl protons. The positional distribution of butyryl groups in isolated positional isomers of butyrate TAG, interesterified TAG mixtures, and natural and interesterified butteroil can be determined by integration of these signals.

Fractionation of the triacylglycerols of lipase-modified butter oil

Triacylglycerols (TGs) of lipase-modified butter oil were separated into saturated, monoene, diene and triene fractions on ap-propylbenzene sulfonic acid solid-phase extraction column loaded with silver ions. Fatty acid analysis of the fractions showed that the amounts of saturated TGs (98.4 mol%) and monoene TGs (26.0 mol%) in the saturated and monoene fractions, respectively, were close to the theoretical amounts of TGs in pure fractions. The column method provides a useful alternative to AgNO3-thin-layer chromatography as a means of separating the TGs of butterfat and producing relatively pure TG fractions for further analysis by gas chromatography (GC) or GC-mass spectrometry.

Separation and identification of neutral cereal lipids by normal phase high-performance liquid chromatography, using evaporative light-scattering and electrospray mass spectrometry for detection.

A novel method was developed for the analysis of molecular species in neutral lipid classes, using separation by normal phase high-performance liquid chromatography, followed by detection by evaporative light-scattering and electrospray ionization tandem mass spectrometry. Monoacid standards, i.e. sterol esters, triacylglycerols, fatty acids, diacylglycerols, free sterols and monoacylglycerols, were separated to baseline on microbore 3 microm-silica gel columns. Complete or partial separation of molecular species in each lipid class permitted identification by automatic tandem mass spectrometry of ammonium adducts, produced via positive electrospray ionization. After optimization of the method, separation and identification of molecular species of various lipid classes was comprehensively tested by analysis of neutral lipids from the free lipid extract of maize flour.

Lipase-catalyzed acidolysis of butterfat with oleic acid: characterization of process and product

The modification of anhydrous butterfat via interesterification reactions with oleic acid catalyzed by a lipase from Mucor circinelloides immobilized by adsorption onto hydrophobic hollow fibers is described. A reasonable degree of incorporation of free (externally added) oleic acid into the triacylglycerols of butterfat has been achieved while short-chain fatty acid residues remained virtually unaffected. Total saturated triacylglycerols decreased by 27%, and triacylglycerols with 32–44 acyl carbons (which contained two or three lauric, myristic, or palmitic acid residues) decreased by 33%. Total monoene and polyene triacylglycerols increased by 21% and 17%, respectively. The triacylglycerols (TAG) of interesterified butterfat had approximately 27% more oleic acid residues and approximately 8% less lauric, 6% less myristic, and 6% less palmitic acid residues than those of the original butterfat; the fraction of low-melting TAG peak increased by 19% whereas that of high-melting TAG decreased by 83%. Although a certain degree of butterfat hydrolysis was observed, enzymatic acidolysis was technically feasible and able to produce a modified butterfat with a stronger nutraceutical character.

Quantitative determination of triacylglycerols separated on capillary columns according to acyl carbon number and level unsaturation

The quantitative determination of triacylglycerols separated according to acyl carbon numbers and level of unsaturation was studied on two 10-m immobilised SE-54 columns. The fat samples were silylated before on-column injection. The 0.2 mm I.D. column showed higher resolving power and the 0.32 mm I.D. column better repeatability. The application to real quantitations was investigated with analyses of butter-fat solid and liquid fractions and of untreated and interesterified 70-30 mixtures of butter-fat solid fraction and hydrogenated rapeseed oil. Changes in the triacylglycerol composition induced by interesterification and fractionation are so pronounced that analysis on capillary columns can be recommended, in spite of only fair repeatability.

Analysis of regioisomers of short-chain triacylglycerols by normal phase liquid chromatography–electrospray tandem mass spectrometry

Abstract The regiospecific distribution of short-chain fatty acids in triacylglycerols (TAGs) influences their physico-chemical, nutritional, and organoleptic properties. This study demonstrates the applicability of normal phase HPLC with positive electrospray ionization mass spectrometry for the identification and structural analysis of regioisomers of short-chain triacylglycerols in nine interesterified mixtures of monoacid triacylglycerols. The finding that only one type of triacylglycerol adduct ion was formed without fragmentation or with minimal fragmentation was critical for liquid chromatography electrospray mass spectrometric quantification and identification of molecular species of TAGs. In the product ion electrospray tandem mass spectrometry, the 4–11 times lower abundance of the ion formed by cleavage of fatty acid from the secondary position relative to that formed by cleavage from the primary positions provided solid basis for differentiation between regioisomers of mono- and dibutyrates. Although the respective differences were lower, mono- and dicaproate regioisomers could be differentiated despite their incomplete separation. The tandem mass spectra of the short-chain triacylglycerols containing three different acyl groups revealed that the regioisomer having the shortest acyl chain in the secondary position, eluted in the first peak. The mixture of the other two regioisomers, where the shortest acyl chain was located in the primary positions, eluted in the second peak. In one instance only did the tandem mass spectra allow to differentiate between the reverse isomers eluting in the second peak. An effective chromatographic resolution of butyrate triacylglycerols and the linear relationship between the molar and area ratios of analyte and standard, enabled the mass spectrometric quantification of their regioisomers.